Original Research published: 22 December 2017 doi: 10.3389/fneur.2017.00704
Borneol attenuates UltrasoundTargeted Microbubble Destructioninduced Blood–Brain Barrier Opening in Focal cerebral ischemia Xiao-guang Zhang 1,2†, Ye Song3†, Chang Shan4†, Xi-fan Wu2, Yan-hua Tong3, Xin-chun Jin5, Wen-lan Liu 6*, Guo-qing Zheng1* and Jie Liu2*
Edited by: Rick Dijkhuizen, University Medical Center Utrecht, Netherlands Reviewed by: Henry Ma, Monash University, Australia Maxime Gauberti, Institut National de la Santé et de la Recherche Médicale (INSERM), France *Correspondence: Wen-lan Liu
[email protected]; Guo-qing Zheng
[email protected]; Jie Liu
[email protected] †
These authors have contributed equally to this work. Specialty section: This article was submitted to Stroke, a section of the journal Frontiers in Neurology
Received: 05 August 2017 Accepted: 06 December 2017 Published: 22 December 2017 Citation: Zhang X-g, Song Y, Shan C, Wu X-f, Tong Y-h, Jin X-c, Liu W-l, Zheng G-q and Liu J (2017) Borneol Attenuates Ultrasound-Targeted Microbubble Destruction-Induced Blood–Brain Barrier Opening in Focal Cerebral Ischemia. Front. Neurol. 8:704. doi: 10.3389/fneur.2017.00704
Frontiers in Neurology | www.frontiersin.org
1 Department of Neurology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China, 2 Translational Center for Stem Cell Research, Tongji Hospital, Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China, 3 Department of Ultrasound, Shanghai Tongji Hospital of Tongji University, Shanghai, China, 4 Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-jin Hospital, Shanghai Jiao-tong University School of Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai, China, 5 Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, The Second Affiliated Hospital of Soochow University, Soochow University, Suzhou, China, 6 The Central Laboratory, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China
Ultrasound-targeted microbubble destruction (UTMD) and the herb medicine borneol can both facilitate the delivery of therapeutic agents to diseased brain regions and serve as promising adjuvant neuroprotective therapies. Our preliminary experiments showed that UTMD could exacerbate ischemic blood–brain barrier (BBB) opening, while borneol can protect the BBB. In this study, we tested the hypothesis that the combination of UTMD and borneol could attenuate UTMD-induced injury to the BBB under ischemic stroke conditions. Male albino mice were subjected to 60-min middle cerebral artery occlusion (MCAO) with reperfusion. Borneol and UTMD was given to mice 3 days before and 24 h after MCAO induction. BBB permeability, brain water contents, ultrastructural changes of the BBB and histopathological alterations were evaluated. Our data demonstrated that UTMD aggravated the leakage of Evans blue dye, ultrastructural alterations of cerebral microvasculature, brain edema, and even induced cerebral hemorrhage in ischemic stroke mice. Pretreatment with borneol significantly attenuated the above detrimental effects of UTMD on the BBB. This study indicates that under ischemic stroke conditions, the BBB becomes vulnerable to UTMD intervention, and the combination of borneol can help to maintain the integrity of the BBB. Keywords: borneol, unfocused, ultrasound-targeted microbubble destruction, ischemic stroke, blood–brain barrier
INTRODUCTION Stroke is the second most common cause of death and the major cause of acquired disability worldwide (1). After several decades of research on neuroprotectants against ischemic stroke, few successful results have been obtained and no single neuroprotectant agent has been approved so far (2). The blood–brain barrier (BBB), a highly selective permeability barrier, separates the circulating blood from the central nervous system (CNS) and plays a pivotal role in maintaining brain homeostasis. BBB opening is frequently seen during cerebral ischemia and reperfusion (3), which could theoretically allow neuroprotectants to get into the affected brain tissue. However, targeted delivery
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Borneol Attenuates UTMD-Induced BBB Opening
humidity of 55 ± 10%. Food and water were available ad libitum. All procedures associated with the care of animals were approved by the Animal Ethics Committee of Shanghai Tongji Hospital and performed according to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, which was published in 1996. All efforts were made to reduce the number of animals used. Animals were randomly divided into four groups: I/R group (n = 16), I/R + borneol group (n = 18), I/R + UTMD group (n = 18), and I/R + borneol + UTMD group (n = 18). Borneol was dissolved in 5% Tween 80 and given to mice by oral gavage at 200 mg/kg 3 days before MCAO induction. UTMD was administered 24 h after MCAO induction.
of neuroprotectants may still be hindered, even by an ischemiainduced “open” BBB (4). Therefore, novel therapeutic strategies that can induce safe and more BBB opening are urgently needed for neuroprotective therapy in ischemic stroke. Mounting evidence from animal studies has demonstrated that ultrasound-targeted microbubble destruction (UTMD) can noninvasively and selectively facilitate the delivery of therapeutic agents to specific brain area through transiently opening the BBB and is increasingly appreciated as a promising adjunct therapy for ischemic stroke (5, 6). Fatar et al. have demonstrated that UTMD significantly reduces the infarct volume and glutamate level in the brain of ischemic rats, supporting its neuroprotective role in acute ischemic stroke (7). Wang et al. reported that transcranial UTMD-mediated vascular endothelial growth factor plasmid delivery significantly reduced apoptosis and infarct size and improved neurologic function in ischemic stroke mice (5). While researchers are obsessed with exploring how UTMD effectively assists the neuroprotective agents in the treatment of CNS diseases, it has also been reported that UTMD’s mechanical and cavitation effects can cause normal brain tissue bleeding (8, 9). Our preliminary experiments also showed that UTMD at a setting that was safe and relatively modest for BBB opening in healthy mice could exacerbate BBB opening and result in erythrocytes extravasations in ischemic stroke mice. These findings raise an important safety concern for the clinical application of UTMD as an adjuvant post-stroke therapy. Herb medicine borneol is a simple bicyclic monoterpene extracted from several species of Artemisia and Dipterocarpaceae. Acting as a good penetration enhancer, borneol remains to be one of the most common Chinese herbal medicine prescribed for treating cerebrovascular disease (10). Animal stroke studies have demonstrated that borneol treatment could effectively alleviate the alterations in BBB integrity including tight junctions (TJs) opening, basal lamina degradation, permeability increase, and brain edema following cerebral ischemia and reperfusion (11, 12). In addition, borneol has been reported to be neuroprotective via anti-apoptosis, scavenging intracellular reactive oxygen species, and maintaining normal mitochondrial membrane potential (13, 14). These multifaceted actions of borneol have prompted us to test the hypothesis that the combination of UTMD and borneol may serve as a safer BBB opening strategy than UTMD alone under ischemic stroke conditions. In this study, we tested the above hypothesis in a mice model of middle cerebral artery occlusion (MCAO) with reperfusion and validate the role of borneol and UTMD in BBB permeability, ultrastructure of microvessels, and brain water content under ischemic states, which might have great significance for clinical treatment of ischemic stroke.
Focal Cerebral Ischemia and Reperfusion Model
Focal cerebral ischemia and reperfusion (I/R) was induced by transient MCAO as previously described (15). Briefly, the mice were anesthetized by inhalation of 5% isoflurane and maintained with 2% isoflurane in a mixture of 70% N2O and 30% O2. Under an operating microscope, right common carotid artery (CCA), internal, and external carotid arteries were exposed through a neck incision. The right CCA was ligatured and the circulation in the right external and internal carotid arteries was temporarily interrupted with a 6-0 silk suture. An arteriotomy was performed in the CCA proximal to the carotid bifurcation. A monofilament nylon suture (Sensas, 80 µm diameter), with its front end coated by “thermomelting” glue, (4 mm long, 190 µm diameter, Jet Melt, Radiospares, Beauvais, France) was introduced via lumen of right CCA advanced into the right internal carotid artery to occlude the origin of the right middle cerebral artery. The filament was withdrawn 60 min after occlusion to permit reperfusion.
Application of Transcranial UTMD
Mice were anesthetized by the intraperitoneal injection of 1% sodium pentobarbital (30 mg/kg). The hair over the skull was removed with an electric clipper and a depilatory cream. For sonification, the head of the mouse was immobilized with a stereotaxic equipment. The ultrasound probe (EC1123, Esaote Medical, Italy) was placed on the water container, between the scalp and the polyurethane membrane filling with ultrasound gel for the reduction of attenuation of ultrasound energy (Figure 1A). The transcranial UTMD was carried out with a diagnostic ultrasound apparatus (MyLabTwice, Esaote Medical, Italy), and simultaneously combined with an intravenous injection of SonoVue ultrasound contrast agent (Bracco) which was mixed with 5 ml normal saline before use. The mean diameter of microbubbles was 2.5 µm and their mean concentration was 5 × 108/ml. The mice were treated with UTMD at the Flash mode (interval for 2 s), sonication frequency of 3.0 MHz, mechanical index (MI) 0.06/1.0, focus depth of 5 cm, volume of microbubble 0.1 ml, and duration 3 min. Throughout the process, the elliptical polyurethane membrane was held vertically in the right hemisphere in order to greater precision of the focus. Contrastenhanced ultrasound perfusion was observed in the ultrasonic real-time angiography images (Figure 1B).
MATERIALS AND METHODS Animals
Male adult Institute of Cancer Research albino mice weighting 25–30 g were obtained from Shanghai Laboratory Animal Research Centre and housed in a temperature-controlled room (22.0 ± 2.0°C) under a 12 h light:12 h dark cycle and relative Frontiers in Neurology | www.frontiersin.org
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Borneol Attenuates UTMD-Induced BBB Opening
FIGURE 1 | Experimental setup (A) and contrast-enhanced ultrasound (CEU) perfusion images of the right hemisphere (B). Arrows show the brain tissue beneath skull. CEU signal increased with time and was mainly concentrated in the microvessels on brain cortex.
Evaluation of BBB Permeability
transmission electron microscopy (TEM), zones of the compromised BBB were cut into small pieces (
